Comparative Glycoproteomic Profiling of Human Body Fluid between Healthy Controls and Patients with Papillary Thyroid Carcinoma

Papillary thyroid carcinoma (PTC) is the most common
type of thyroid
cancer among women worldwide. It is confirmed mainly by fine-needle
aspiration biopsy (FNAB), an invasive diagnostic method. The key proteins
responsible for thyroid hormone biosynthesis are glycosylated. Hence,
changes in site-specific glycosylation are associated with thyroid
cancer. Integrated quantitative proteomic and glycoproteomic analyses
of body fluids from patients with PTC may identify potential noninvasive
biomarkers, improve diagnostic accuracy, and elucidate the basic mechanisms
of tumor development. In the present study, we demonstrate an integrated,
highly reproducible, rapid method involving body fluid proteome and
glycoproteome analysis. Moreover, this method may quantitatively profile
protein glycosylation. Intact N-glycopeptides from
the urine and plasma of healthy controls (HC), PTC, and PTC with Hashimoto’s
thyroiditis (PHT) were enriched by hydrophilic interaction liquid
chromatography. Sialic acid was removed from the N-glycopeptides with trifluoroacetic acid and heat. The desialo-N-glycopeptides were analyzed by HCD-MS/MS using stepped
collision energies and several search engines for quantitative profiling.
Ninety-two altered proteins and 134 intact N-glycopeptides
were isolated from the plasma and urine samples of the three groups
(90 samples from 15 subjects). To the best of our knowledge, this
study is the first to compare the plasma and urinary proteomes and
glycoproteomes of HC, PTC, and PHT. Moreover, we reveal a novel indicator
(ratio of fucosylated to nonfucosylated N-glycopeptide
or F/NF) through desialo-N-glycopeptide analysis.
These differently expressed glycoproteins and F/NF may serve as biomarkers
contributing to clinical cancer diagnostics and could be used to improve
diagnostic accuracy noninvasively.